The national crop germplasm is at risk due to unregulated entry of infected plant materials and to our own mono-culture breeding practices that have lead to the shrinking of our genetic base. The United States Department of Agriculture (USDA) has already identified 2,000 exotic plant pathogens of which 550 have been recognized as posing a threat to US agriculture. One such plant parasite is Phakospora pachirhzi,the causative agent of soybean rust. This fungus is native to Asia and has already spread to Africa and to South America, thus posing a significant new threat to the soybean industry both in the United States and abroad. To date, none of our commercially important soybean cultivars have been bred for rust resistance. Indeed, there have been harvest losses due to sensitivity to this fungus in other countries.
Thus, there is a need for a breeding program that would assist in the rapid development of resistant lines against pathogens introduced either by nature, accident or bio-terrorism. The present invention provides a solution to this problem as it provides a robust DNA marker-assisted breeding protocol that allows for rapid development of new lines faster than with traditional breeding programs. Specifically, in vitro flowering allows seed harvest in approximately three months from the time of explant implantation in the tissue culture media, thus enabling, for example, four cycles of soybean production annually.
The methods of the present invention are also especially useful for the incorporation of desirable agronomic traits into plants, including for example, resistance to cold and heat, drought, salt, water stress, insects, pathogens and disease by gene cloning or through DNA marker assisted breeding.
The methods of the present invention are also especially useful for the incorporation of genes that can uptake heavy metals, and accumulate nutrients and trace elements, from contaminated soils, a process referred to as phytoremediation. Phytoremediation employs plants to remediate contaminated soils, typically either by phytostabilization or by phytoextraction. With phytostabilization, plants are used to stabilize contaminated soils by decreasing wind and water erosion as well as decreasing water infiltration and contaminant leaching into groundwater. Phytoextraction attempts to remove contaminants are from the rhizosphere through plant uptake and the contaminants are accumulated in roots, leaves and/or stems. The plant materials are then harvested and the contaminants reclaimed from the plant biomass or the materials are disposed of at a hazardous waste facility.
The methods of the present invention are also especially useful for the production of human interest proteins (“HIP”s) in plants as compared to animal systems. HIPs cover a broad range of commercially important, value-added products that include vaccines, antibodies, hormones, peptides, cytokinins, and enzymes. HIP-based technologies are broad based in their economic impact on greenhouse and farm economy, land value as well as the pharmaceutical industry. By using plants as efficient bio-reactors and synthesizing pharmaceutical product on an acre scale, costs can be reduced for drugs, supplements and food additives.
The completion of the sequencing of the human genome has driven pharmaceutical companies around the world to significantly increase their spending on research and development. In 2001,the pharmaceutical industry spent 30 billion dollars alone on drug design. This figure represents a 19% increase over that spent in 2000.
As reported by the Pharmaceutical Research and Manufacturers of America, more than 1,000 drugs are in clinical studies or are awaiting final approval from the Food and Drug Administration. Of these, 400 address cancer therapies, 200 for special needs children, 100 each for heart disease and stroke, 26 for Alzheimer's disease, 25 for diabetes, 19 for arthritis, 16 for Parkinson's disease and 14 for osteoporosis. Collectively the industry must be prepared to accept the inevitable pressure of designing manufacturing systems that will control drug costs. Today state-of-the-art production of genetically engineered proteins is through mammalian cell culture. A minimum investment of $100 million is needed to build a factory that will produce a mere couple of hundred grams of product annually. As this does not usually generate sufficient quantities, third world countries will face ensuing hardships as they often cannot afford the startup costs and/or lack a sufficient number of trained personnel to produce their own medications.
Moreover, the use of mammalian cell culture as bio-reactors for HIP production carries with it certain intrinsic health risks. Specifically, an inherent danger of viral contamination associated with mammalian-derived materials necessitates exhaustive safety testing and validation of production processes. Animals infected with certain zoonotic viruses have transmitted fatal illnesses to humans. Numerous mouse-derived cell lines contain endogenous retroviruses and some demonstrate species-specific tumorigenic potential. Oncogenic xenotropic murine retroviruses are of particular concern because of the many theoretical risks they present to humans. New viruses with altered pathogenicity or host range could be generated through genetic recombination. Tumors also may form through integration of the viral genome in close proximity to a host oncogene, thus activating the oncogene.
Thus, given the issues above, the plant biotechnology sector has a great interest in expressing mammalian proteins in plants in a way that would allow their commercial exploitation. The advantages of producing therapeutic recombinant proteins in plants are many. These include the ability to fabricate HIP production on an agricultural scale, which significantly lowers manufacturing costs. Further, one may possibly transport highly sought and needed therapeutic proteins that remain stable in dry seed for extended periods of time. Most importantly, no human or animal pathogens have ever been reported that have the ability to infect plants. Thus, viral contamination that is observed in animal cell culture is absent in plants.
Already, transgenic plants have been produced to express a number of different HIP molecules using a variety of plant species. See e.g. Mason and Amtzen, Trends in Biotechnology 13:388-392 (1995); Arakawa et al., Nature Biotechnology 16:282-297 (1998); Mor et al., Trends in Microbiology 449-453 (1998); Ma et al., Nature Medicine 4:601-605 (1998); Zeitland et al., Nature Biotechnology 16: 1361-1364 (1998). Transgenic potato plants are producing HIP that are responsive to diabetes, and to cholera (a disease that affects five million people annually and kills 200,000) and to enterotoxigenic Escherichia coli (ETEC), the leading cause of diarrhea in children under five in third world nations. ETEC pathology is profound and results in 650 million cases of diarrhea that kills 800,000 children annually (Block, 1986).
In tobacco, a surface protein from Streptococcus mutans is being synthesized that should confer passive immunity with respect to tooth decay. Additionally, tobacco is being used to produce a second vaccine against Hepatitis B, an infectious disease that annually cripples two billion people. Unfortunately, the levels of gene expression that have been observed in tobacco are low and often disturbingly variable. See also Daniell et al., Trends in Plant Sciences 5:219-226 (2001).
Despite the advance in the production of HIP in plants, issues remain to be resolved. For example, differences in HIP production have not only been delineated among plants of different cultivars but also among plants from the same cultivars. Low output and variable gene expression is not the only problem that is encountered in these production systems. Some plants are easy to engineer, but produce HIP that cannot be ingested or easily purified. For example, the leaves of tobacco contain toxic alkaloids and therefore cannot be eaten. Alternatively, attempts to purify proteins from transgenic tobacco leaves also are compromised due to the abundant phenolic contamination. Similarly, the utility of transgenic potatoes is limited as the raw tuber is not especially palatable. The amounts needed to ingest a therapeutically active dose would be difficult to tolerate. Moreover, the average potato contains only two percent protein of which the HIP is likely to be a minor component.
In contrast, some transgenic plants like tomato and banana are easily ingested, however, their utility as sources of edible HIP is compromised by the fact that the amount of protein found in these fruits is low, which no doubt limits the amount of HIP made. Furthermore, banana transformation rates are low and each transgenic fruiting banana plant requires a minimum of two years from the time of genetic manipulation to harvest.
Grains, such as corn, are more suitable bioreactors than bananas. Specifically, palatable seed can be easily produced in large numbers using relatively unsophisticated farming techniques. Unfortunately, corn has several major limitations. The amount of protein/seed is low and growing sufficient amounts in contained quarters would be difficult. Further, transgenic corn pollen travels on average 600 feet, and would pose containment problems that are significantly reduced using other plants.
Thus, there remains a need for a robust alternative HIP bio-reactor technology where speed of delivery is linked to high quantity protein production and problems associated with pollen containment are drastically reduced. The present invention satisfies this need through the production of transgenic plants in a contained environment through a novel in vitro flowering method. The present invention provides speed of delivery linked to high quantity protein production as well as drastically reducing problems and costs associated with pollen containment issues.
Although, in vitro flowering has been previously observed in capsicum, bamboo and in orchids (Yu and Goh, Plant Physiology,vol. 123, 1325-1336 (2003); Bodhipadma and Leung, In Vitro Cellular and Developmental Biology Plant 39(5) September-October 2003, 536-539 (2003); and Ho and Chang “In Vitro Flowering of Albino Bamboo (Bambusa Oldhamnii Munro) Regenerants Derived from an Eleven-Year Old Embryogenic Cell Line” 2003 ISHS Acta Horticulturae 461: International Symposium on Biotechnology of Tropical and Subtropical Species Part 2 (2003)), there remains a need for in vitro flowering methods that produce viable seeds from the flowers. In these previous in vitro flowering experiments, the in vitro flowers were induced in tissue culture from intervening stem or modified stem-like structure, but failed to produce viable seed.